AJP - GI Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 290: G293-G300, 2006. First published September 15, 2005; doi:10.1152/ajpgi.00275.2005
0193-1857/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/2/G293    most recent
00275.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cottrell, J. J.
Right arrow Articles by Burrin, D. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cottrell, J. J.
Right arrow Articles by Burrin, D. G.

MUCOSAL BIOLOGY

Glucagon-like peptide-2 protects against TPN-induced intestinal hexose malabsorption in enterally refed piglets

J. J. Cottrell,1 B. Stoll,1 R. K. Buddington,2 J. E. Stephens,1 L. Cui,1 X. Chang,1 and D. G. Burrin1

1United States Department of Agriculture-Agricutural Research Service, Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas; and 2Department of Biological Sciences, Mississippi State University, Starkville, Mississippi

Submitted 15 June 2005 ; accepted in final form 13 September 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Premature infants receiving chronic total parenteral nutrition (TPN) due to feeding intolerance develop intestinal atrophy and reduced nutrient absorption. Although providing the intestinal trophic hormone glucagon-like peptide-2 (GLP-2) during chronic TPN improves intestinal growth and morphology, it is uncertain whether GLP-2 enhances absorptive function. We placed catheters in the carotid artery, jugular and portal veins, duodenum, and a portal vein flow probe in piglets before providing either enteral formula (ENT), TPN or a coinfusion of TPN plus GLP-2 for 6 days. On postoperative day 7, all piglets were fed enterally and digestive functions were evaluated in vivo using dual infusion of enteral (13C) and intravenous (2H) glucose, in vitro by measuring mucosal lactase activity and rates of apical glucose transport, and by assessing the abundances of sodium glucose transporter-1 (SGLT-1) and glucose transporter-2 (GLUT2). Both ENT and GLP-2 pigs had larger intestine weights, longer villi, and higher lactose digestive capacity and in vivo net glucose and galactose absorption compared with TPN alone. These endpoints were similar in ENT and GLP-2 pigs except for a lower intestinal weight and net glucose absorption in GLP-2 compared with ENT pigs. The enhanced hexose absorption in GLP-2 compared with TPN pigs corresponded with higher lactose digestive and apical glucose transport capacities, increased abundance of SGLT-1, but not GLUT-2, and lower intestinal metabolism of [13C]glucose to [13C]lactate. Our findings indicate that GLP-2 treatment during chronic TPN maintains intestinal structure and lactose digestive and hexose absorptive capacities, reduces intestinal hexose metabolism, and may facilitate the transition to enteral feeding in TPN-fed infants.

total parenteral nutrition; porcine; sodium glucose transporter-1; glucose transporter-2

TOTAL PARENTERAL NUTRITION (TPN) is widely used in the clinical management of infants with gastrointestinal disorders that are unable to tolerate enteral feeding. Whereas TPN is a life saving therapy, the provision of enteral nutrients is an important stimulus for intestinal growth, and prolonged TPN can result in intestinal atrophy (29), thereby reducing digestive capacity of the intestine through reductions in villus height and brush-border digestive enzymes (27). Because TPN has been observed to reduce intestinal lactase (9, 27) and glucose transport activity (21, 26), it has been suggested that TPN may increase the risk of malabsorption, resulting in necrotizing enterocolitis (25) and osmotic imbalances (39). Additionally, TPN reduces intestinal blood flow and alters the metabolism of the intestinal mucosa, including decreased protein synthesis and increased glucose metabolism (5, 7, 14, 31). Given that most preterm infants receive some degree of TPN before commencement of enteral feeding, it is conceivable that TPN may compromise the transition to full enteral feeding.

Glucagon-like peptide-2 (GLP-2) is a gut hormone that is posttranslationally processed from the proglucagon gene product localized in enteroendocrine L cells in response to enteral nutrition, especially carbohydrate and lipid (13, 20, 28, 36). A robust intestinal trophic response to GLP-2 treatment has been observed in many studies due in part to stimulation of epithelial cell survival, crypt cell proliferation, and protein synthesis (2, 6, 8, 12, 17, 40). GLP-2 may be useful in the clinical management of TPN fed neonates, because it has been approved for treatment of adult short-bowel syndrome and many of its biological actions counteract the negative effects of TPN. Increased villus height after GLP-2 treatment is accompanied by increased intestinal disaccharidase and peptidase expression and activity (2). Moreover, transient increases in basolateral glucose net uptake have been observed in GLP-2-treated rodents and in TPN-fed piglets (17). The GLP-2-mediated stimulation of glucose uptake in rodents has been linked to increased intestinal abundance of sodium glucose transporter-1 (SGLT-1) in the brush-border membrane (BBM) (10).

It was previously shown (5) that chronic TPN induces hexose malabsorption in vivo in neonatal piglets and that this was associated with mucosal villus atrophy and reduced intestinal blood flow and lactase activity. We also observed that chronic TPN resulted in increased intestinal lactate release, indicative of increased mucosal glycolytic metabolism. Thus, given previous evidence of the intestinal trophic and vasoactive actions of GLP-2, we hypothesized that GLP-2 treatment of TPN-fed piglets would prevent mucosal atrophy and maintain normal intestinal lactase activity and hexose absorptive function, facilitating the transition from TPN to enteral nutrition. The dose of GLP-2 used in this study was selected based on previous evidence that it produced a robust intestinal trophic response and supraphysiological plasma GLP-2 concentration in TPN-fed piglets (6). Moreover, the current dose used also corresponds to the pharmacological GLP-2 dose used in a recently published clinical study with short-bowel patients (23). Therefore, the aim of this experiment was to quantify intestinal lactose digestion and hexose metabolism in piglets nourished on chronic TPN or TPN plus GLP-2 infusion for 6 days. To quantify the metabolic fate of intestinal glucose metabolism, we used a dual infusion of enteral (13C) and intravenous (2H) glucose, respectively, and further characterized the mucosal and cellular determinants of glucose transport, including SGLT-1 and glucose transporter-2 (GLUT-2) abundance.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Animals and experimental design. Neonatal crossbred piglets (Large White x Hampshire x Duroc) were acquired from the Texas Department of Criminal Justice (Huntsville, TX) at 4 days of age. Piglets were fed enterally for 7 days with 50 g/kg body wt sow milk formula (Litter Life; Merrick, Middleton, WI), which consisted of the following: 527 g lactose, 100 g fat, and 250 g protein. The protocol was approved by the Animal Care and Use Committee of the Baylor College of Medicine and was conducted in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals.

The surgical procedure used in this experiment has been described previously (5, 38). In summary, after overnight food withdrawal, catheters were surgically inserted into the carotid artery, jugular vein, portal vein, and duodenum. Additionally, ultrasonic flow probes (6S-8S, Transonics, Ithaca, NY) were implanted on the portal vein at 11 days of age. All piglets received TPN for 24 h during surgical recovery, after which piglets were assigned to one of the following treatments: enteral formula (ENT; n = 4), continuous intravenous infusion of TPN via the jugular vein (TPN; 240 ml·kg–1·day–1, n = 10), or TPN plus coinfusion of GLP-2 (500 pmol·kg–1·h–1, n = 9) for 7 days. Human GLP-2 was mixed in sterile 0.9% NaCl with 0.1% human serum albumin vehicle (American Peptide). Piglets were weighed daily, and feed intake was adjusted accordingly. The TPN solution consisted of an elemental diet of free amino acids, dextrose, lipid (Intralipid, Baxter Healthcare, Deerfield, IL), vitamins A, D, and E (Vital E, Schering-Plough Animal Health, Kenilworth, NJ), multivitamin solution (Multitrace-5, American Reagent Laboratories, Shirley, NY), B vitamins (Vedco, St. Joseph, MO), and folic acid (Vet. Med. Rx, Houston, TX) (8). The macronutrient and fluid intake of all three experimental groups was ~240 ml, 13 g protein, and 900 kJ–1·kg–1·day–1.

Enteral feeding and glucose tracer protocol. After 6 days of treatment (18 days age), all piglets were given a primed (10 ml/kg) continuous intraduodenal infusion (10 ml·kg–1·h–1) of formula for 6 h. Administration of TPN and GLP-2 was stopped 6 h before intraduodenal infusion, and formula-fed piglets were fasted overnight. Paired carotid arterial and portal venous blood samples (3 ml) were collected the day before intraduodenal feeding and at 3, 4, 5, and 6 h postfeeding. The hematocrit, hemoglobin (Hemocue, Angelholm, Sweden) and blood O2 and CO2 (Chiron Diagnostics, Halstead, Essex, UK) were immediately analyzed, the remaining blood was centrifuged at 1,100 g and 4°C for 10 min, and was plasma removed. All samples were frozen in liquid nitrogen and stored at –80°C until analysis.

At the end of the 6-h intraduodenal feeding, piglets were euthanized by an intravenous barbiturate overdose (Beutanasia-D, Schering-Plough Animal Health). The small intestine from the ligament of Treitz to colon was removed via a midline laparotomy, then immediately placed on an ice-cool metal surgical pan and flushed with 50 ml ice-cold 0.9% NaCl. The undigested intestinal and stomach contents and saline flush were collected and weighed, and the intestine was cut in half. The proximal half (jejunum) and distal half (ileum) were measured and weighed. Ten-centimeter lengths of proximal jejunum and ileum were removed for determination of active and passive glucose sequestration, and four small intestinal segments were fixed in 10% buffered formalin at 4°C for 24 h, then stored in 70% EtOH at 4°C for morphometric analysis. The remaining intestine was frozen for analysis of protein, DNA, SGLT-1, and GLUT-2 abundance and lactase activity.

Gas chromatography-mass spectrometry. Gas chromatography-mass spectrometry (GCMS) was performed on the pentaacetate derivative of glucose and pentafluorobenzyl bromide derivative of lactate using a 5890 Series II gas chromatograph linked to a 5890 series quadrupole mass spectrometer (Hewlett Packard, Palo Alto, CA). The isotopic enrichment (IE) was determined using electron impact ionization for ions with a mass-to-charge ratio of 242–244 and 131–134 for [13C]glucose or [2H]glucose and [13C]lactate, respectively. 13CO2 was measured using continuous-flow gas flow coupled to an isotope ratio mass spectrometer (Gasbench II coupled to DELTAplusXL, Thermofinnigan).

Plasma and tissue analyses. Plasma glucose and lactate were measured specrophotometrically (Spectramax 190, Molecular Dynamics) using enzyme-based assays (ThermoDMA, Louisville, CO and Trinity Biotech, Wicklow, Ireland, respectively). Plasma galactose concentrations were measured using an electrochemical analyzer (2300 Stat Plus, Yellow Springs Instruments, Yellow Springs, OH). Protein, DNA, and lactase activity were measured on tissue homogenized in phosphate-buffered saline (pH 7.4) as described previously (4, 14). Morphometric analyses of formalin-fixed, paraffin-embedded sections were performed after hematoxylin and eosin staining. Villus height, area, crypt depth, and muscularis thickness were measured using an Axiophot microscope (Carl Zeiss, Göttingen, Germany and Scion image beta 4.0.2 software, National Institutes of Health, Bethesda, MD). Abundance of GLUT-2 (Chemicon International) and SGLT-1 (Alpha Diagnostics) at ~60 and 57 kDa, respectively, in whole mucosa, and isolated BBM extracts was determined via Western blot analysis and subsequent densitometric analysis (Personal Densitometer SI, Molecular Dynamics, and PDSI Scanner Control v5.03 software, Amersham Biosciences, Buckinghamshire, UK). Western blot analysis inlays presented in Figs. 3 and 4 were generated by scanning films from each Western blot (Expression 636, Epson, Nagano, Japan) and cropping bands from ENT, TPN, and GLP-2 piglets that were representative of the treatment mean (Photoshop version 7, Adobe Systems). BBM tissue was prepared as per previously established methods, after centrifugation in 10 mM MgCl2 at 2,400; 19,000; and 39,000 g, providing a minimum 10-fold enrichment of lactase activity (15, 30).



View larger version (26K):
[in this window]
[in a new window]
  		Fig. 3. Jejunum (A) and ileum (B) brush-border membrane (BBM) and mucosal sodium glucose transporter-1 (SGLT-1) abundance in piglets given ENT, TPN, or TPN + GLP-2 for 6 days and then refed enterally for 6 h. Means ± SE, the nos. of animals per group were enteral (4), TPN (10), and TPN + GLP-2 (9). Different superscripts indicate statistical differences between treatment for BBM and mucosa based on analysis of variance and Tukey's test (P < 0.05). Differing superscripts are used to denote statistical treatment differences for BBM (a, b) and mucosa (x, y). Ileum mucosal SGLT-1 was not reliably detectable and is not shown.

 


View larger version (30K):
[in this window]
[in a new window]
  		Fig. 4. Jejunal (A) and ileum (B) BBM GLUT-2 abundance in piglets given ENT, TPN, or TPN + GLP-2 for 6 days and then refed enterally for 6 h. Means ± SE, the nos. of animals per group were enteral (4), TPN (10), and TPN + GLP-2 (9). Different superscripts indicate statistical differences between treatment for BBM and mucosa based on analysis of variance and Tukey's test (P < 0.05). Differing superscripts are used to denote statistical treatment differences for BBM (a, b) and mucosa (x, y).

 
In vitro apical glucose uptake. The segments from each region were everted, and 1-cm sleeves were secured by silk ligatures on stainless steel rods (0.5-cm diameter). Throughout the process, the segments and mounted sleeves were kept in cold (2–4°C), aerated (95% O2 with 5% CO2) Ringer solution. Beginning 45 min after death, the sleeves were first incubated for 5 min in 37°C aerated ringers before they were transferred for 2 min in 37°C aerated ringers containing 0.2, 1, 5, 25, or 50 mM unlabeled glucose. After the incubation, the sleeves were rinsed for 20 s in cold, glucose-free Ringer solution. Tracer concentrations of [14C]D-glucose and [3H]L-glucose (American Radiolabelled Chemicals) were added to the incubation solutions to respectively quantify the amount of D-glucose associated with the active tissues and to correct for D-glucose associated with the adherent fluid, and they were passively absorbed independent of carriers. After the rinse, the sleeves were removed from the rods, weight recorded, solubilized (Solvable, Perkin Elmer, Boston, MA), and scintillant was added (Ultima Gold, Perkin Elmer) for determination of radioactivity by liquid-scintillation counting (Tricarb 2500, Perkin Elmer). Rates of glucose uptake, which represent apical transport, were expressed as nanomoles of D-glucose accumulated per minute per milligram of wet intestinal tissue (nmol·mg–1·min–1) (3). Total intestinal lactose digestive capacity and apical glucose transport capacities were calculated as the product of lactase-specific activities and maximum rates of glucose uptake (Vmax) x intestinal mass normalized to body weight (g/kg).

Calculations. The net portal balance (NPB) of glucose and galactose was calculated using the difference in the portal (Cport) and arterial (Cart) concentrations and portal blood flow (PBF) (1). In the calculations of portal glucose and lactate kinetics, PBF was converted to plasma flow by correcting for the hematocrit. The NPB represents the net intestinal absorption of glucose and galactose into the portal blood. Intestinal O2 uptake was calculated as per Ref. 2. Intestinal lactate production was also calculated as per Ref. 2 by substituting arterial and portal lactate concentrations.

(1)

(2)
Tracer [13C]glucose uptake kinetics were determined by incorporating the IE in mole percent atomic excess (MPE) into Eq. 1 as shown in Eq. 3. Utilization of arterial [2H]glucose in second-pass metabolism was calculated by the arterial-portal difference (4). Intestinal [13C]lactate and 13CO2 production were calculated as per Eq. 4, substituting to concentrations and IE of [13C]lactate and 13CO2, respectively.

(3)

(4)
Whole body flux (Q) in µmol·kg–1·h–1 was calculated from the rate of tracer infusion (R) and enrichments of the infusate (IEinfusate) and plasma (IEplasma) (5).

(5)

Statistical analyses. All data were tested for significance using a general linear model ANOVA and a Kruskal-Wallis nonparametric analysis to confirm normality (Minitab 13, Minitab, PA). Means were considered significantly different at the <0.05 confidence interval, and superscripts, where present, indicate differences in means (P < 0.05) as indicated by a post hoc Tukey's test. Rates of apical glucose uptake at the different glucose concentrations were subjected to nonlinear regression analysis (Enzfit, Biosoft, Elsevier) to estimate maximum rates of transport (Vmax) and apparent affinity constants (Km*).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
As observed in previous experiments, TPN resulted in lower values for intestinal weight, jejunal and ileal villus height, and protein and DNA concentration compared with ENT, and this remained evident even after 6 h of enteral refeeding (Table 1). In accordance with its trophic properties, administering GLP-2 resulted in higher intestinal mass, jejunum and ileum villus height, and protein and DNA content after 6 h of refeeding compared with TPN. However, the level of GLP-2-mediated protection against TPN did not represent a return to values observed in ENT-fed piglets. Lactase-specific activity was reduced by TPN, compared with ENT, whereas infusion of GLP-2 resulted in intermediate activity but not significantly different from ENT or TPN. Piglets receiving GLP-2 had higher total intestinal lactose digestive capacity than those receiving TPN alone, but activity was significantly less than for the ENT-fed piglets. The treatment differences were due mainly to the variation in intestinal mass rather than specific activity.


View this table:
[in this window]
[in a new window]
  		Table 1. Intestinal weight, protein and DNA contents, and lactase activities in neonatal piglets fed for 7 days with enteral, TPN, or TPN with GLP-2 and then fed enterally for 6 h

 
Fasted basal PBF was not different among treatments (3.42, 4.68, and 2.67 for ENT, TPN, and GLP-2, P = 0.15). The only difference for the degree of feeding hyperemia averaged across the 6-h refeeding period was the higher value for TPN compared with the GLP-2 piglets, but neither group was different from enteral (Table 2). Intestinal O2 uptake was lower for piglets receiving TPN relative to ENT, irrespective of GLP-2 infusion. Intestinal CO2 production was lowest in TPN piglets and highest for ENT piglets. Values were intermediate for GLP-2 treatment but not significantly different to ENT or TPN.


View this table:
[in this window]
[in a new window]
  		Table 2. Portal blood flow, oxygen uptake, and carbon dioxide production in neonatal piglets fed for 7 days with enteral, TPN, or TPN with GLP-2 for 7 days, then fed enterally for 6 h

 
The intraduodenal infusion of formula provided equivalent lactose and hence glucose intake to all treatment groups (3.95, 4.03, 3.95 mM; P = 0.58). After the 6-h enteral refeeding protocol, in all treatments, <1% of the lactose and glucose provided was recovered in the contents of the stomach and intestinal saline flush, but this does not include lactose and glucose undigested in the small intestine that entered the colon.

Arterial and portal glucose concentrations measured hourly between 3 and 6 h after intraduodenal formula infusion were not different among treatments (Table 3). However, glucose NPB was highest in ENT-fed piglets, comprising 93% of intake. Values for TPN and GLP-2 were lower (27% and 58% of intake, respectively), suggesting both groups had incomplete digestion and absorption of the administered lactose. Arterial galactose concentrations were increased by GLP-2 compared with ENT and TPN, and this was independent of concomitant increases in portal galactose concentrations, which did not differ from those of ENT piglets. The lowest portal galactose concentrations were measured in TPN-treated piglets (P < 0.05 compared with ENT and GLP-2 piglets). Galactose NPB was ~55% of intake in ENT-fed piglets, considerably <93% of intake for glucose absorption. Although this may seem low, this is higher than 38% observed for ENT-fed piglets in a prior experiment (5). TPN reduced galactose NPB to 27% of dietary intake, whereas GLP-2 improved galactose uptake approximately twofold (52%), which was comparable with ENT-fed piglets.


View this table:
[in this window]
[in a new window]
  		Table 3. Plasma concentrations and net portal balances of glucose, galactose and lactate in neonatal piglets fed enterally, with TPN, or TPN with GLP-2 infusion for 7 days, then fed enterally for 6 hours

 
TPN resulted in a higher arterial lactate concentration compared with ENT, whereas GLP-2-treated piglets were intermediate and did not differ from ENT and TPN piglets. Similarly portal vein plasma lactate concentrations were elevated in piglets receiving TPN but less so when GLP-2 was administered. Net portal release of lactate was lowest in ENT-fed piglets, highest in TPN-fed piglets, and intermediate for GLP-2 piglets. Thus mucosal glycolysis was elevated in TPN and, to a lesser degree, GLP-2 groups, which was confirmed by [13C]lactate kinetics.

Due to a small sample size and some sample error, it was not possible to calculate in vivo [13C]glucose and [2H]glucose tracer kinetics for ENT-fed piglets. Comparisons of in vivo [13C]glucose and [2H]glucose tracer kinetics are restricted to the TPN and GLP-2 treatments (Table 4). Treatment effects were not detected for arterial and portal [13C]glucose IE and concentrations, despite trends of lower portal IE and arterial concentrations in GLP-2-infused piglets. Likewise, [13C]glucose absorption, utilization, or whole body flux did not differ between TPN and GLP-2 piglets. However, arterial and portal [13C]lactate enrichment and concentrations and net portal [13C]lactate production were significantly higher in TPN piglets (Table 5), suggesting TPN alone elevated intestinal glycolysis. As per intestinal CO2 production, 13CO2 production was not different between TPN and GLP-2. To discriminate between first pass metabolism of [13C]glucose and metabolism of arterial glucose by the PDV, intravenous coinfusion of [2H]glucose was performed (Table 5). GLP-2 infusion increased whole body [2H]glucose flux, which is consistent with the increased intestinal capacities to absorb glucose (see below). Second-pass glucose metabolism did not appear to be affected by administering GLP-2, because portal [2H]glucose utilization and extraction of intravenous [2H]glucose did not differ between TPN and GLP-2 piglets.


View this table:
[in this window]
[in a new window]
  		Table 4. Rates of [13C]glucose and [2H]glucose whole body flux and gut absorption and utilization kinetics in neonatal piglets fed either enterally, TPN, or TPN with GLP-2 infusion for 7 days and then fed enterally for 6 h

 

View this table:
[in this window]
[in a new window]
  		Table 5. Rates of portal [13C]lactate, and 13CO2 kinetics in neonatal piglets fed enterally, with TPN, or TPN with GLP-2 infusion for 7 days, then fed enterally for 6 h

 
The Vmax for apical glucose uptake in the jejunum was highest in ENT piglets, whereas values for TPN pigs were reduced by approximately two thirds after chronic TPN (Fig. 1). GLP-2 increased the Vmax compared with TPN, but values remained lower than ENT. This pattern was also apparent in the ileum, with the exception that the protective effect of GLP-2 on Vmax was not apparent. The treatment differences in Vmax were independent of changes in the Km*. Total intestinal lactose digestive capacities of ENT piglets (Fig. 2) exceeded the lactose intake by more than fivefold. The excess capacity was reduced in TPN piglets to about twofold, with GLP-2 piglets having an intermediate capacity relative to intake (3.5-fold). Similarly, maximum glucose absorptive capacities were highest for ENT piglets, but when total hexose intake was considered (glucose + galactose), the capacity exceeded intake by only 50%. Although glucose transport capacity of TPN piglets (4.05 mmol·kg–1·h–1) was equivalent to lactose intake (3.95 mmol·kg–1·h–1), it represented only half of the combined intake of hexoses (7.9 mmol·kg–1·h–1). Despite stimulating a higher glucose transport capacity relative to TPN piglets, providing GLP-2 did not result in capacities that exceeded total hexose intake.



View larger version (12K):
[in this window]
[in a new window]
  		Fig. 1. In vitro maximal intestinal glucose transport activity (Vmax) in piglets given enteral nutrition (ENT), total parenteral nutrition (TPN), or TPN + glucagon-like peptide-2 (GLP-2) for 6 days and then refed enterally for 6 h. Means ± SE, the nos. of animals per group were enteral (4), TPN (8), and TPN + GLP-2 (7). Different superscripts indicate statistical differences between treatment for jejunum and ileum based on analysis of variance and Tukey's test (P < 0.05).

 


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 2. Estimated mucosal lactose digestive and glucose transport capacities in piglets given ENT, TPN, or TPN + GLP-2 for 6 days and then refed enterally for 6 h. Means ± SE, the nos. of animals per group were ENT (4), TPN (8), and TPN + GLP-2 (7). Different superscripts indicate statistical differences between treatment for jejunum and ileum based on analysis of variance and Tukey's test (P < 0.05).

 
Despite differences in Vmax and glucose transport capacity, abundances of SGLT-1 detected by Western blotting in the jejunum BBM and mucosa did not differ between TPN and ENT piglets (Fig. 3). However, BBM and mucosa SGLT-1 abundance was higher in GLP-2 compared with TPN piglets. SGLT-1 was not reliably detected in mucosal homogenates from the ileum (data not shown). TPN significantly reduced ileum BBM SGLT-1 abundance compared with ENT-fed piglets, with an intermediate abundance for GLP-2 piglets. Jejunum and ileum mucosal GLUT-2 abundance did not differ among treatments (Fig. 4). Jejunum BBM prepared from TPN and GLP-2 piglets had 10-fold higher abundances of GLUT-2 compared with ENT piglets. GLUT-2 abundances in the BBM prepared from the ileum were lower for GLP-2 compared with ENT and TPN piglets.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 DISCLOSURES
 REFERENCES
 
The functional and metabolic disturbances during chronic TPN and refeeding are associated with reduced intestinal glucose absorption, protein synthesis, and blood flow (9). This led us to postulate that reduced hexose absorption after chronic TPN occurred via a combination of either reduced hexose transporter abundance or increased mucosal glucose metabolism. The current study was designed to test whether a pharmacological dose of GLP-2, which prevents TPN-induced mucosal atrophy, translates into improved intestinal glucose absorption in piglets during refeeding. The dose of GLP-2 used in this study was selected based on previous evidence that it produced a robust intestinal trophic response and supraphysiological plasma GLP-2 concentration in TPN-fed piglets (6). Consistent with previous experiments, TPN induced mucosal atrophy and reduced intestinal weight, villus height, villus area, protein and DNA content, and lactose digestive capacity (5, 22, 29, 31). Our results also indicate that the protective effect of GLP-2 was demonstrated by improvement in all of these parameters compared with TPN-fed piglets. Our results indicate that GLP-2 treatment was able to partially maintain in vivo intestinal hexose absorptive function in TPN-fed piglets. Moreover, we show that the GLP-2-induced increase in hexose absorptive capacity occurred via increased villus surface area and upregulation of intestinal glucose transport and reduced intestinal glycolytic metabolism.

Lactose hydrolysis in TPN-fed piglets was significantly lower than in ENT-fed piglets, suggesting that the TPN-induced mucosal atrophy causes functional defects in lactose digestion (5). GLP-2 treatment during TPN maintained lactase-specific activity and hence digestive capacity, consistent with other findings for lactase and other BBM disaccharides in piglets and mice (2, 33–35). Moreover, the GLP-2 treatment maintained lactose digestive capacity at a level twice that of TPN-fed piglets and approximately four times higher that the lactose intake during refeeding. The lactose digestive capacity was greatest for ENT piglets being approximately eight times higher than the lactose intake. Consistent with the estimated excess lactose digestive capacities, the lactose recovery from the stomach and intestine was <1%, with similarly low values for GLP-2 and ENT piglets. Thus, although we did not account for lactose that could have passed into the colon or lost via mild diarrhea during the 6-h refeeding period, the low recovery of lactose is congruent with rapid digestion. Interestingly, lactase activity is considered to be the limiting factor for lactose digestion in adults (11, 16, 32), whereas our findings indicate lactase activity of pigs is in excess, even for those maintained by TPN. This likely reflects the developmentally high lactose digestive capacity in neonates (32).

One of the principal findings of this experiment was that GLP-2 resulted in higher absorption of glucose and galactose during the refeeding period compared with TPN. As observed previously (5), chronic TPN markedly reduced in vivo glucose absorption to ~30% of intake; this was only one-third the rate (90%) found in ENT-fed piglets. Infusion of GLP-2 partially maintained glucose uptake at ~58% of intake, yet this was still less than ENT. The net rate of intestinal glucose absorption is determined by the combined processes of apical mucosal transport and mucosal metabolism. We previously reported that TPN increased intestinal glucose metabolism to lactate, reducing glucose appearance in the portal vein. In this study, the simultaneous infusion of enteral (13C) and intravenous (2H) glucose isotopes during the refeeding period allowed us to determine that intestinal production of [13C]lactate was halved in GLP-2-infused piglets compared with piglets receiving TPN alone. Furthermore, utilization of intravenous [2H]glucose in second-pass metabolism was considerably less (<10% of intake) compared with first-pass utilization of enteral [13C]glucose ({approx}66% of intake). These findings indicate that enterally absorbed glucose was the principal source of glucose metabolized during the refeeding of the TPN and GLP-2 treatments. Although there was no difference in [13C]glucose absorption, it is noteworthy that GLP-2 increased the [2H]glucose whole body flux. This is most likely due to an increase in glucose absorption in GLP-2-treated piglets, rather than an increase in endogenous glucose release, because the increase in whole body [2H]glucose flux with GLP-2 treatment (~1.2 mmol·kg–1·h–1) was largely accounted for by increased glucose absorption ({approx}1.0 mmol·kg–1·h–1). Collectively, the NPB of glucose and increased [2H]glucose flux indicate that GLP-2 treatment increased in intestinal glucose absorption and reduced intestinal glycolytic metabolism of glucose.

The findings for in vivo glucose absorption were consistent with apical glucose transport capacities calculated from in vitro measurements, with both showing that capacities were lowest for TPN, intermediate for GLP-2, and highest for ENT piglets. Apical glucose transport capacities measured in this experiment represent a combination of the activities of apical transporters and villus surface area. Even after 6 h of refeeding, TPN piglets had lower in vitro rates of glucose uptake in the jejunum and, in conjunction with intestinal villus atrophy, resulted in glucose uptake capacity that was equivalent to glucose intake during the refeeding period. However, the uptake capacity would not have been sufficient for the absorption of the combined glucose and galactose (hexose) load released from the dietary lactose. Although the decline in jejunum glucose uptake was prevented by GLP-2 treatment, as previously reported (37), this protective effect did not extend to the ileum. Therefore the improvement in glucose transport capacity of GLP-2 compared with TPN piglets is due to a combination of improved villus structure, increasing the mucosal absorptive surface area, and increased jejunum glucose transport, presumably due to increased abundance of mucosal glucose transporters.

Absorption of glucose across the BBM is dependent on two or more carriers. SGLT-1 is a low-capacity, high-affinity mucosal glucose and galactose transporter that is primarily responsible for apical glucose transport at low concentrations. GLUT-2 is a high-capacity, low-affinity hexose transporter in the basolateral membrane that is responsible for the transport of glucose out of the enterocyte into the blood. GLUT-2 is now recognized to be transiently recruited to the apical membrane where it can account for a significant proportion of apical glucose transport during the processing of meals (18, 19, 24). Although the relative contributions of SGLT-1 and GLUT-2 to the measured rates of uptake were not determined, we observed an increase in the Vmax for mucosal glucose uptake with GLP-2 treatment. Furthermore, the different Vmaxs were independent of changes in Km*. This suggests the treatment differences for rates of uptake were due to differences in the abundances of the apical membrane glucose transporters.

The lower glucose uptake by the proximal small intestine of TPN compared with GLP-2 piglets coincided with a lower BBM abundance of SGLT-1, but not GLUT-2. A similar pattern was observed in the distal segment, except for the lower abundance of GLUT2 in the whole tissue of GLP-2 piglets. Although TPN resulted in lower BBM abundances of SGLT-1 in both intestinal regions compared with ENT piglets, GLUT-2 abundances were not affected. These findings suggest the abundances of SGLT-1 and GLUT-2 in the BBM are not regulated in parallel. Moreover, because GLUT-2 translocates to the BBM in response to high luminal glucose concentrations, it is conceivable that the relative differences in GLUT-2 abundance observed in this experiment are a result of the capacity of SGLT-1 to reduce luminal glucose concentrations (24). Assuming that increased BBM abundance equates to increased transport capacity, the increased BBM SGLT-1 abundance in ENT piglets provided an increased capacity for removal of luminal glucose and thereby removed the trigger for GLUT-2 trafficking to the BBM. In contrast, in TPN-fed piglets, the inherently lower SGLT-1 abundance would result in lower glucose absorption and thereby lead to increased luminal glucose concentrations and provide a stimulus for GLUT-2 trafficking to the BBM. However, our observations with GLP-2 treatment do not fit this model, in that BBM abundance of both SGLT-1 and GLUT-2 increased. This can be explained in part by the observation in separate reports that GLP-2 increases trafficking of either SGLT-1 or GLUT-2 into the BBM, independent of luminal glucose concentrations (1, 10), but this is the first evidence that SGLT-1 and GLUT-2 translocation occurs concurrently.

In summary, the current study provides novel in vivo evidence that the intestinal trophic effects of GLP-2 treatment during TPN translate into improved intestinal function. We found that chronic GLP-2 treatment during 6 days TPN improved in vivo glucose and galactose absorption during 6 h of refeeding. This was attributed to the ability of GLP-2 to maintain intestinal villus surface area, increase lactose digestive and apical transport capacities of hexoses in addition to reduced intestinal glycolytic metabolism. Although poor gastric emptying and motor function contribute to feeding intolerance in premature infants, the transition to enteral feeding is limited by poor intestinal digestion and glucose absorption. Thus these findings provide support for future clinical studies in infants to assess whether GLP-2 treatment during TPN improves intestinal digestion and absorptive function, thereby accelerating the transition to enteral feeding and reducing the time to full feeding.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
DISCLOSURES
 REFERENCES
 
This work was supported by National Institutes of Health Grant HD-33920 (to D. G. Burrin) and by the USDA-ARS under Cooperative Agreement Number 58-6250-6-001.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
REFERENCES
 
The contents of this publication do not necessarily reflect the views or policies of the U.S. Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.


   ACKNOWLEDGMENTS
 
The authors thank M. Riedijk for assistance during the experiment and X. Guan and B. Nichols for helpful discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. G. Burrin, USDA-ARS, Children's Nutrition Research Center, Dept. of Pediatrics, Baylor College of Medicine, Houston, TX 77030 (e-mail: dburrin{at}bcm.tmc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 

  1. Au A, Gupta A, Schembri P, and Cheeseman CI. Rapid insertion of GLUT2 into the rat jejunal brush-border membrane promoted by glucagon-like peptide 2. Biochem J 367: 247–254, 2002.[CrossRef][Web of Science][Medline]
  2. Brubaker PL, Izzo A, Hill M, and Drucker DJ. Intestinal function in mice with small bowel growth induced by glucagon-like peptide-2. Am J Physiol Endocrinol Metab 272: E1050–E1058, 1997.[Abstract/Free Full Text]
  3. Buddington RK, Elnif J, Puchal-Gardiner AA, and Sangild PT. Intestinal apical amino acid absorption during development of the pig. Am J Physiol Regul Integr Comp Physiol 280: R241–R247, 2001.[Abstract/Free Full Text]
  4. Burrin D, Guan X, Stoll B, Petersen YM, and Sangild PT. Glucagon-like peptide 2: a key link between nutrition and intestinal adaptation in neonates? J Nutr 133: 3712–3716, 2003.[Abstract/Free Full Text]
  5. Burrin DG, Stoll B, Chang X, Van Goudoever JB, Fujii H, Hutson SM, and Reeds PJ. Parenteral nutrition results in impaired lactose digestion and hexose absorption when enteral feeding is initiated in infant pigs. Am J Clin Nutr 78: 461–470, 2003.[Abstract/Free Full Text]
  6. Burrin DG, Stoll B, Guan X, Cui L, Chang X, and Holst JJ. Glucagon-like peptide 2 dose-dependently activates intestinal cell survival and proliferation in neonatal piglets. Endocrinology 146: 22–32, 2005.[CrossRef][Web of Science][Medline]
  7. Burrin DG, Stoll B, Jiang R, Chang X, Hartmann B, Holst JJ, Greeley GH Jr, and Reeds PJ.. Minimal enteral nutrient requirements for intestinal growth in neonatal piglets: how much is enough? Am J Clin Nutr 71: 1603–1610, 2000.[Abstract/Free Full Text]
  8. Burrin DG, Stoll B, Jiang R, Petersen Y, Elnif J, Buddington RK, Schmidt M, Holst JJ, Hartmann B, and Sangild PT. GLP-2 stimulates intestinal growth in premature TPN-fed pigs by suppressing proteolysis and apoptosis. Am J Physiol Gastrointest Liver Physiol 279: G1249–G1256, 2000.[Abstract/Free Full Text]
  9. Castillo RO, Pittler A, and Costa F. Intestinal maturation in the rat: the role of enteral nutrients. JPEN J Parenter Enteral Nutr 12: 490–495, 1988.[Abstract/Free Full Text]
  10. Cheeseman CI. Upregulation of SGLT-1 transport activity in rat jejunum induced by GLP-2 infusion in vivo. Am J Physiol Regul Integr Comp Physiol 273: R1965–R1971, 1997.[Abstract/Free Full Text]
  11. Dahlqvist A and Thomson DL. The digestion and absorption of lactose by the intact rat. Acta Physiol Scand 61: 20–33, 1964.[Web of Science][Medline]
  12. Drucker DJ, Erlich P, Asa SL, and Brubaker PL. Induction of intestinal epithelial proliferation by glucagon-like peptide 2. Proc Natl Acad Sci USA 93: 7911–7916, 1996.[Abstract/Free Full Text]
  13. Dube PE and Brubaker PL. Nutrient, neural and endocrine control of glucagon-like peptide secretion. Horm Metab Res 36: 755–760, 2004.[CrossRef][Web of Science][Medline]
  14. Dudley MA, Wykes LJ, Dudley AW Jr, Burrin DG, Nichols BL, Rosenberger J, Jahoor F, Heird WC and Reeds PJ. Parenteral nutrition selectively decreases protein synthesis in the small intestine. Am J Physiol Gastrointest Liver Physiol 274: G131–G137, 1998.[Abstract/Free Full Text]
  15. Fan MZ, Adeola O, McBurney MI, and Cheeseman CI. Kinetic analysis of L-glutamine transport into porcine jejunal enterocyte brush-border membrane vesicles. Comp Biochem Physiol A 121: 411–422, 1998.[CrossRef][Medline]
  16. Gray GM and Santiago NA. Disaccharide absorption in normal and diseased human intestine. Gastroenterology 51: 489–498, 1966.[Web of Science][Medline]
  17. Guan X, Stoll B, Lu X, Tappenden KA, Holst JJ, Hartmann B, and Burrin DG. GLP-2-mediated up-regulation of intestinal blood flow and glucose uptake is nitric oxide-dependent in TPN-fed piglets. Gastroenterology 125: 136–147, 2003.[CrossRef][Web of Science][Medline]
  18. Helliwell PA, Richardson M, Affleck J, and Kellett GL. Regulation of GLUT5, GLUT2 and intestinal brush-border fructose absorption by the extracellular signal-regulated kinase, p38 mitogen-activated kinase and phosphatidylinositol 3-kinase intracellular signalling pathways: implications for adaptation to diabetes. Biochem J 350: 163–169, 2000.[CrossRef][Web of Science][Medline]
  19. Helliwell PA, Richardson M, Affleck J, and Kellett GL. Stimulation of fructose transport across the intestinal brush-border membrane by PMA is mediated by GLUT2 and dynamically regulated by protein kinase C. Biochem J 350: 149–154, 2000.[CrossRef][Web of Science][Medline]
  20. Holst JJ, Christiansen J, and Kuhl C. The enteroglucagon response to intrajejunal infusion of glucose, triglycerides, and sodium chloride, and its relation to jejunal inhibition of gastric acid secretion in man. Scand J Gastroenterol 11: 297–304, 1976.[Web of Science][Medline]
  21. Inoue Y, Espat NJ, Frohnapple DJ, Epstein H, Copeland EM, and Souba WW. Effect of total parenteral nutrition on amino acid and glucose transport by the human small intestine. Ann Surg 217: 604–612, 1993.[Web of Science][Medline]
  22. Johnson LR, Copeland EM, Dudrick SJ, Lichtenberger LM, and Castro GA. Structural and hormonal alterations in the gastrointestinal tract of parenterally fed rats. Gastroenterology 68: 1177–1183, 1975.[Web of Science][Medline]
  23. Jungersten L, Ambring A, Wall B, and Wennmalm A. Both physical fitness and acute exercise regulate nitric oxide formation in healthy humans. J Appl Physiol 82: 760–764, 1997.[Abstract/Free Full Text]
  24. Kellett GL and Helliwell PA. The diffusive component of intestinal glucose absorption is mediated by the glucose-induced recruitment of GLUT2 to the brush-border membrane. Biochem J 350: 155–162, 2000.[CrossRef][Web of Science][Medline]
  25. Kien CL. Digestion, absorption, and fermentation of carbohydrates in the newborn. Clin Perinatol 23: 211–228, 1996.[Web of Science][Medline]
  26. Kien CL, McClead RE, and Cordero L Jr. In vivo lactose digestion in preterm infants. Am J Clin Nutr 64: 700–705, 1996.[Abstract/Free Full Text]
  27. Kotler DP, Levine GM, and Shiau YF. Effects of luminal nutrition and metabolic status on in vivo glucose absorption. Am J Physiol Gastrointest Liver Physiol 240: G432–G436, 1981.[Abstract/Free Full Text]
  28. Larsson LI, Holst J, Hakanson R, and Sundler F. Distribution and properties of glucagon immunoreactivity in the digestive tract of various mammals: an immunohistochemical and immunochemical study. Histochemistry 44: 281–290, 1975.[CrossRef][Web of Science][Medline]
  29. Levine GM, Deren JJ, Steiger E, and Zinno R. Role of oral intake in maintenance of gut mass and disaccharide activity. Gastroenterology 67: 975–982, 1974.[Web of Science][Medline]
  30. Maenz DD and Patience JF. L-Threonine transport in pig jejunal brush border membrane vesicles. Functional characterization of the unique system B in the intestinal epithelium. J Biol Chem 267: 22079–22086, 1992.[Abstract/Free Full Text]
  31. Niinikoski H, Stoll B, Guan X, Kansagra K, Lambert BD, Stephens J, Hartmann B, Holst JJ, and Burrin DG. Onset of small intestinal atrophy is associated with reduced intestinal blood flow in TPN-fed neonatal piglets. J Nutr 134: 1467–1474, 2004.[Abstract/Free Full Text]
  32. O'Connor TP and Diamond J. Ontogeny of intestinal safety factors: lactase capacities and lactose loads. Am J Physiol Regul Integr Comp Physiol 276: R753–R765, 1999.[Abstract/Free Full Text]
  33. Petersen YM, Burrin DG, and Sangild PT. GLP-2 has differential effects on small intestine growth and function in fetal and neonatal pigs. Am J Physiol Regul Integr Comp Physiol 281: R1986–R1993, 2001.[Abstract/Free Full Text]
  34. Petersen YM, Elnif J, Schmidt M, and Sangild PT. Glucagon-like peptide 2 enhances maltase-glucoamylase and sucrase-isomaltase gene expression and activity in parenterally fed premature neonatal piglets. Pediatr Res 52: 498–503, 2002.[CrossRef][Web of Science][Medline]
  35. Petersen YM, Hartmann B, Holst JJ, Huerou-Luron I, Bjornvad CR, and Sangild PT. Introduction of enteral food increases plasma GLP-2 and decreases GLP-2 receptor mRNA abundance during pig development. J Nutr 133: 1781–1786, 2003.[Abstract/Free Full Text]
  36. Polak JM, Bloom S, Coulling I, and Pearse AG. Immunofluorescent localization of enteroglucagon cells in the gastrointestinal tract of the dog. Gut 12: 311–318, 1971.[Abstract/Free Full Text]
  37. Sangild PT, Petersen YM, Schmidt M, Elnif J, Petersen TK, Buddington RK, Greisen G, Michaelsen KF, and Burrin DG. Preterm birth affects the intestinal response to parenteral and enteral nutrition in newborn pigs. J Nutr 132: 3786–3794, 2002.[Medline]
  38. Stoll B, Burrin DG, Henry J, Yu H, Jahoor F, and Reeds PJ. Substrate oxidation by the portal drained viscera of fed piglets. Am J Physiol Endocrinol Metab 277: E168–E175, 1999.[Abstract/Free Full Text]
  39. Worley G, Claerhout SJ, and Combs SP. Hypophosphatemia in malnourished children during refeeding. Clin Pediatr (Phila) 37: 347–352, 1998.[Abstract/Free Full Text]
  40. Yusta B, Estall J, and Drucker DJ. Glucagon-like peptide-2 receptor activation engages bad and glycogen synthase kinase-3 in a protein kinase A-dependent manner and prevents apoptosis following inhibition of phosphatidylinositol 3-kinase. J Biol Chem 277: 24896–24906, 2002.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
D. W. Nelson, S. G. Murali, X. Liu, M. C. Koopmann, J. J. Holst, and D. M. Ney
Insulin-like growth factor I and glucagon-like peptide-2 responses to fasting followed by controlled or ad libitum refeeding in rats
Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2008; 294(4): R1175 - R1184.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
D. G. Burrin, B. Stoll, X. Guan, L. Cui, X. Chang, and D. Hadsell
GLP-2 rapidly activates divergent intracellular signaling pathways involved in intestinal cell survival and proliferation in neonatal piglets
Am J Physiol Endocrinol Metab, January 1, 2007; 292(1): E281 - E291.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/2/G293    most recent
00275.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cottrell, J. J.
Right arrow Articles by Burrin, D. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cottrell, J. J.
Right arrow Articles by Burrin, D. G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.